Phosphonic acids represent a potent, yet underexploited, group of bioactive compounds with great promise in the treatment of human disease. A wide variety of phosphonates are produced in nature and many have useful bioactive properties. Importantly, the biological targets of phosphonic acids vary substantially, allowing them to be used for treating a variety of health conditions. In our initial funding period we showed that phosphonate biosynthesis is surprisingly common in nature and that a wealth of uncharacterized natural products await characterization. Armed with these results, we are now ready to move fully into the discovery and development phase of the project. In the next five years we expect to characterize a large number of novel phosphonate compounds. Their chemical structures will be determined, their bioactivity profiles assessed and their biosynthetic pathways elucidated. We will use this information to develop strains that efficiently and economically produce the most useful candidates. The proposed Program Project addresses each of these topics via a multidisciplinary research program involving microbiology, biochemistry, chemistry, metabolic engineering and structural biology. We propose four intertwined research projects to discover, design and develop novel and known phosphonic acid antibiotics. The first project involves discovery, sequencing and characterization of gene clusters encoding phosphonic add biosynthesis using genomics, microbial genetics and molecular biology. The second project is focused on structural elucidation of the wealth of new compounds that have been discovered in the current funding period and on the biochemical reconstruction of the biosynthetic pathways of phosphonic acid antibiotics. The third project will focus on structural biology and enzymology of unusual catalysts involved in phosphonate biosynthesis and the various resistance determinants that may be utilized to overcome the biological activities of phosphonates. The fourth project will employ cutting-edge synthetic biology approaches to engineer both natural and designed phosphonic acid biosynthetic pathways for economical production of medically and industrially important phosphonic acid compounds. Each of the four projects will be aided by an Analytical Core resource that will provide modern mass-spectrometry and nuclear magnetic resonance instrumentation and technical support. The entire project will be housed in the new Institute for Genomic Biology at the University of Illinois, where the program project team occupies a single, large contiguous, laboratory.